Functionalized derivatives of hyaluronic acid, formation of hydrogels in situ using same, and methods for making and using same

ABSTRACT

Methods for chemical modification of hyaluronic acid, formation of amine or aldehyde functionalized hyaluronic acid, and the cross-linking thereof to form hydrogels are provided. Functionalized hyaluronic acid hydrogels of this invention can be polymerized in situ, are biodegradable, and can serve as a tissue adhesive, a tissue separator, a drug delivery system, a matrix for cell cultures, and a temporary scaffold for tissue regeneration.

TECHNICAL FIELD OF THE INVENTION

This invention is directed to biomaterials for spatially and temporallycontrolled delivery of bioactive agents such as drugs, growth factors,cytokines or cells. In particular, this invention teaches versatilemethods for chemical crosslinking of high molecular weight hyaluronicacid under physiological conditions in situ, to form polymerizablebiodegradable materials. The methods are based on the introduction offunctional groups into hyaluronic acid (HA) via formation of an activeester at the carboxylate of the glucuronic acid moiety as anintermediate and subsequent substitution with a side chain containing anucleophilic group on one end and a (protected) functional group on theother end. The introduced functional groups allow for crosslinking ofthe HA derivatives. Crosslinked hyaluronic acid hydrogels of thisinvention are useful in various surgical applications and as a temporaryscaffold for tissue regeneration, e.g., in cartilage repair.

BACKGROUND OF THE INVENTION

Repair of Articular Cartilage

The failure of regenerating persistent hyaline cartilage by surgicalprocedures has prompted investigators to attempt repair using biologicalstrategies. The biological repair of articular cartilage is, with a fewexceptions, still at an experimental stage. Biological cartilage repairhas been approached in two basic ways. First, autologous chondrocyteshave been transplanted into a lesion to induce repair (Grande et al., J.Orthop. Res. 7, 208–214 (1989); Brittberg et al., New Engl. J. Med. 331,889–895 (1994); Shortkroffet al., Biomaterials 17, 147–154 (1996)).Chondrocytes may be obtained from a low-loaded area of a joint andproliferated in culture (see Grande; Brittberg; Shortkroff, supra), ormesenchymal stem cells may be harvested, e.g., from the iliac crestmarrow, and induced to differentiate along the chondrocyte lineage usinggrowth factors (Harada et al., Bone 9, 177–183 (1988); Wakitani et al.,J. Bone Joint Surg. 76-A, 579–592 (1994)). The chondrocytetransplantation procedures currently attempted clinically, althoughpromising, are hampered because technically they are very challenging,the cell preparation is very expensive, and the potential patient poolis limited by age, defect location, history of disease, etc. Cells havealso been transplanted into cartilage defects in the form ofperichondral grafts, e.g., obtained from costal cartilage, but withlimited success due to the limit in donor material and the complicationof endochondral ossification of the graft site observed in longtermfollow-up (Amiel et al., Connect. Tissue Res. 18, 27–39 (1988);O'Driscoll et al., J. Bone Joint Surg. 70-A, 595–606 (1988); Homminga etal., Acta Orthop. Scand. 326–329 (1989); Homminga et al., J. Bone JointSurg. 72-B, 1003–1007 (1990)). A second approach is aimed at therecruitment of mesenchymal stem cells from the surrounding connectivetissue, e.g., synovium, using chemotactic and/or mitogenic factors(Hunziker and Rosenberg, J. Bone Joint Surg. 78-A, 721–733 (1996); seealso U.S. Pat. No. 5,368,858). The availability of growth factors andcytokines in recombinant form and the lack of complicated celltransplantation make this procedure a very attractive alternative. Theshortcoming of both procedures is the difficulty to stably anchor therepair-inducing factors, whether tissue grafts, cells, or growthfactors, within the defect site. Also, outlining of the space that is tobe repaired, e.g., by filling it with a matrix material, appears to becrucial to recreate a level cartilage surface (Hunziker and Rosenberg,supra). Thus far, the availability of candidate matrix materials hasbeen the limiting factor, and anchoring of materials seeded withchondrocytes and/or chondrogenic factors difficult, explaining theunsatisfactory results obtained with currently available materials suchas polylactic acid and polyglycolic acid scaffolds (Freed et al., J.Biomed. Mat. Res. 28, 891–899 (1994); Chu et al., J. Biomed. Mat. Res.29, 1147–1154 (1995)); calcium phosphate minerals (Nakahara et al.,Clin. Orthop. 276, 291–298 (1992)), fibrin sealants (Itay et al., Clin.Orthop. 220, 284–303 (1987)), and collagen gels (Wakitani et al., J BoneJoint Surg. 71-B, 74–80 (1989)). We have developed novel biodegradablematerials based on hyaluronic acid which are optimized for thebiological requirements posed on a repair material in a synovial jointand which allow in situ polymerization.

Biology of Hyaluronic Acid and its Therapeutic Use

Hyaluronic acid (HA) is unique among glycosaminoglycans in that it isnot covalently bound to a polypeptide. HA is also unique in having arelatively simple structure of repeating nonsulfated disaccharide unitscomposed of D-glucuronic acid (GIcUA) and N-acetyl-D-glucosamine(GIcNAc) (Scott et al., The Chemistry, Biology and Medical Applicationsof Hyaluronan and Its Derivatives, T. C. Laurent (ed.), Portland Press,London, (hereinafter “Hyaluronan and Its Derivatives”), pp. 7–15(1998)). Its molecular mass is typically several million Daltons. HA isalso referred to as hyaluronan or hyaluronate, and exists in severalsalt forms (see formula I).

HA is an abundant component of cartilage and plays a key structural rolein the organization of the cartilage extracellular matrix as anorganizing structure for the assembly of aggrecan, the large cartilageproteoglycan (Laurent and Fraser, FASEB J. 6, 2397–2404 (1992); Mörgelinet al., Biophys. Chem. 50, 113–128 (1994)). The noncovalent interactionsof aggrecan and link protein with HA lead to the assembly of a largenumber of aggrecan molecules along the HA-chain and mediate retention ofaggrecan in the tissue. The highly negatively charged aggrecan/HAassemblies are largely responsible for the viscoelastic properties ofcartilage by immobilizing water molecules. A number of cell surfacereceptors for HA have been described and shown to play a critical rolein the assembly of the pericellular matrix of chondrocytes and othercells, e.g., isoforms of CD44 and vertebrate homologues of Cdc37(Knudson and Knudson, FASEB J. 7, 1233–1241 (1993); Grammatikakis etal., J. Biol. Chem. 270, 16198–16205 (1995)), or to be involved inreceptor-mediated endocytosis and degradation of HA to control HA levelsin tissues and body fluids (Laurent and Fraser, supra; Fraser et al.,Hyaluronan and Its Derivatives, pp. 85–92 (1998)). Blocking of theinteraction of these receptors with HA in prechondrogenic micromasscultures from embryonic limb bud mesoderm inhibits chondrogenesis,indicating that the establishment and maintainance of a differentiatedchondrocyte phenotype is at least in part dependent on HA andHA-receptor interactions (Maleski and Knudson, Exp. Cell. Res. 225,55–66 (1996)).

HA and its salts are currently being used in therapy for arthropathiesby intraarticular injection (Strachnan et al., Ann. Rheum. Dis. 49,949–952 (1990); Adams, Hyaluronan and Its Derivatives, pp. 243–253(1998)), in opthalmic surgery for intraocular lens implantation(Denlinger, Hyaluronan and Its Derivatives, pp. 235–242 (1998), topromote wound healing in various tissues (King et al., Surgery 109,76–84 (1991)), or more recently, in derivatized and/or crosslinked formto manufacture thin films which are used for tissue separation (forreview see Laurent and Fraser, supra; Weiss, Hyaluronan and ItsDerivatives, pp. 255–266 (1998); Larsen, Hyaluronan and Its Derivatives,pp. 267–281 (1998); Band, Hyaluronan and Its Derivatives, pp. 33–42(1998)). Extensive efforts have been made by various laboratories toproduce derivatives of HA with unique properties for specific biomedicalapplications. Most of the developments have been focusing on theproduction of materials such as films or sponges for implantation andthe substitution of HA with therapeutic agents for delayed releaseand/or prolonged effect (for review see Band, supra; Prestwich et al.,Hyaluronan and Its Derivatives, pp. 43–65 (1998); Gustafson, Hyaluronanand Its Derivatives, pp. 291–304 (1998)). Strategies have includedesterification of HA (U.S. Pat. Nos. 4,957,744 and 5,336,767),acrylation of HA (U.S. Pat. No. 5,410,016), and cross-linking of HAusing divinyl sulfone (U.S. Pat. No. 4,582,865) or glycidyl ether (U.S.Pat. No. 4,713,448). However, the modified HA molecules show alteredphysical characteristics such as decreased solubility in water and/orthe chemical reaction strategies used are not designed for crosslinkingof HA under physiological conditions (in an aqueous environment, at pH6.5–8.0). It is well known that polyaldehydes can be generated byoxidizing sugars using periodate (Wong, CRC Press, Inc., Boca Rayton,Fla., pp. 27 (1993); European Patent No. 9615888). Periodate treatmentoxidizes the proximal hydroxyl groups (at C2 and C3 carbons ofglucuronic acid moiety) to aldehydes thereby opening the sugar ring toform a linear chain (Scheme 1). While periodate oxidation allows for theformation of a large number of functional groups, the disadvantage isthe loss of the native backbone structure. Consequently, the generatedderivative may not be recognized as HA by cells. In fact, hydrogelsformed by using periodate oxidized HA as a crosslinker, e.g., incombination with the HA-amines described herein, showed very limitedtissue transformation and poor cellular infiltration in the rat ectopicbone formation model (FIG. 6). This is in sharp contrast to theHA-aldehyde derivatives described herein.

The introduction of free amino groups on HA, which could be used forfurther convenient coupling reactions under mild physiologicalconditions, has been a subject of great interest. Previous methods haveproduced a free amino group on high molecular weight HA by alkalineN-deacetylation of its glucosamine moiety (Curvall et al., Carbohvdr.Res. 41, 235–239 (1975); Dahl et al., Anal. Biochem. 175, 397–407(1988)). However, concomitant degradations of HA via beta-elimination inthe glucuronic acid moiety was observed under the harsh reactionconditions needed. This is of particular concern because low molecularweight HA fragments, in contrast to high molecular weight HA, have beenshown to be capable of provoking inflammatory responses (Noble et al.,Hyaluronan and Its Derivatives, pp. 219–225 (1998)). An early reportclaimed that carbodiimide-catalyzed reaction of HA with glycine methylester, a monofunctional amine, led to the formation of an amide linkage(Danishefsky and Siskovic, Carbohydr. Res. 16, 199–201 (1971)). Thishowever, has been proven by a number of studies not to be the case (Kuoet al., Bioconjugate Chem. 2, 232–241 (1991); Ogamo et al., Carbohydr.Res. 105, 69–85 (1982)). Under mildly acidic conditions the unstableintermediate O-acylisourea is readily formed, which in the absence ofnucleophiles, rearranges by a cyclic electronic displacement to a stableN-acylurea (Kurzer and Douraghi-Zedeh, Chem. Rev. 67, 107–152 (1967)).This O→N migration of the O-acylisourea also occurs when the nucleophileis a primary amine (Kuo et al., supra) and any amide formation that doesoccur is insignificant as reported by Ogamo et al., supra. Experimentswhere high molecular weight HA (Mr˜2×10⁶ Da) was reacted with an excessof the fluorescent label 5-aminofluorescine in the presence of thecarbodiimide EDC achieved only 0.86% of theoretical labelling. Theintroduction of a terminal hydrazido group on HA with a variable spacerhas recently been achieved and has led to the ability to conduct furthercoupling and crosslinking reactions (Pouyani and Prestwich, BioconjugateChem. 5, 339–347 (1994), U.S. Pat. Nos. 5,616,568, 5,652,347, and5,502,081; Vercruysse et al., Bioconjugate Chem. 8, 686–694 (1997)).

It is an objective of this invention to provide a method for moreversatile modification of HA with various functional groups that allowfor crosslinking of the HA derivatives under physiological conditions.It is another objective that the method of functionalization does notcompromise the molecular weight or chemical identity (except of thetarget carboxyl group for coupling) of HA. It is a further objectivethat the method of functionalization provides HA molecules that are welltolerated in vivo and are biodegradable.

It is also an objective of this invention to identify HA derivatives andmethodology for in situ polymerization thereof to provide abiodegradable scaffold for tissue regeneration. It is another objectivethat the HA materials can be polymerized in the presence of cells toserve as a vehicle for cell transplantation. It is a further objectiveto provide methodology for functionalization and cross-linking of HAthat allows for variations in the biomechanical properties of the formedgels as well as in the sensitivity to cellular infiltration anddegradation.

SUMMARY OF THE INVENTION

Biomaterials for spatially and temporally controlled delivery ofbioactive agents such as drugs, growth factors, cytokines or cells, area key factor for tissue repair. In particular, in situ polymerizablebiodegradable materials are needed for cartilage resurfacing that aredesigned to withstand the mechanical forces in a joint. We havedeveloped a versatile method for chemical crosslinking of high molecularweight hyaluronic acid under physiological conditions. The method isbased on the introduction of functional groups into hyaluronic acid byformation of an active ester at the carboxylate of the glucuronic acidmoiety and subsequent substitution with a side chain containing anucleophilic group on one end and a (protected) functional group on theother end. We have formed hyaluronic acid with amino or aldehydefunctionality, and formed hydrogels with modified hyaluronic acid andbifunctional crosslinkers or mixtures of hyaluronic acid carryingdifferent functionalities using active ester- or aldehyde-mediatedreactions. Physical and chemical properties of the hydrogels of thisinvention were evaluated using biomechanical testing, and by assayingsensitivity towards degradation by glycosidases such as testicularhyaluronidase. Biocompatibility was evaluated using cell culture assaysand subcutaneous implantation of the hyaluronic acid materials in rats.This in vivo assay is also the established model for induction ofectopic bone formation by members of the transforming growth factor βfamily (TGF-β), and several crosslinked hyaluronic acid materials ofthis invention gave excellent ectopic bone formation in vivo when loadedwith appropriate growth factor(s).

As set forth below in the detailed description of the invention, thecompositions of the invention have many therapeutic uses. For example,compositions of the invention may be used to stem hemorrhage in generalsurgery, reconstruct nerves and vessels in reconstructive, neuro- andplastic surgery, and to anchor skin, vascular, or cartilage transplantsor grafts in orthopedic, vascular, and plastic surgery. Compositions ofthe invention are also useful as vehicles for the delivery of cells orbioactive molecules such as growth factors to stimulate focal repair.Local delivery of growth factors facilitates wound healing and tissueregeneration in many situations, not only in promoting bone formationand stimulating cartilage repair in orthopedic procedures, but also,e.g., in treating pathological wound conditions such as chronic ulcers.These compositions may also serve as a scaffold to generate artificialtissues through proliferation of autologous cells in culture. On theother hand, the anti-adhesive property of some compositions with respectto cells render such compositions particularly suitable to generatetissue separations and to prevent adhesions following surgery. Theviscoelastic properties of HA make it particularly well suited for thispurpose, and it is used clinically to achieve temporal pain relief byrepeated intraarticular injections in arthropathies as a “jointlubricant”, as a protective agent for eye irritations and in ophthalmicsurgery, as a barrier to cells in facial and other reconstructions inplastic surgery and dentistry, in reconstructive surgery of tendons, insurgical procedures in the urogenital system, and in thoracic surgery.The injectable nature of the compositions of the invention also rendersthem suitable for tissue augmentation in plastic surgery, where the HAmatrix serves primarily as an inert biocompatible filler material(Balasz and Laurent, Hyaluronan and Its Derivatives, pp. 325–326(1998)), e.g., for filling dermal creases or lip reconstruction.

HA hydrogels match several of the desired properties for a biodegradablematerial biocompatible with cells. The relatively simple repetitivestructure of HA allows for specific modification and introduction of alarge number of functional groups, for crosslinking to generatehydrogels with excellent physical proprties. HA hydrogels have alsosuccessfully been used as a delivery vehicle in chondrocytetransplantation studies (Robinson et al., Calcif Tissue Int. 46, 246–253(1990)) and HA has proven its biocompatibility in various forms inclinical practice (for review see Laurent and Fraser, supra; Balazs andLaurent, supra).

The reaction mechanisms we have explored for in situ polymerization ofHA derivatives are compatible with an aqueous environment and arenon-toxic to cells. The aldehyde-mediated crosslinking strategies followreactions occurring physiologically in crosslinking of fibrillarcollagens and elastin. NHS-esters provide an alternative for rapidformation of stable bonds under physiological conditions, primarily byreaction with primary amines. The technology of NHS-ester-mediatedprotein crosslinking has been developed for materials with applicationsin plastic surgery that require in situ polymerization (U.S. Pat. No.5,413,791)).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a ninhydrin test after reductive alkylationof HA and HA-aldehyde in the presence of putrescine. Reductivealkylation was carried out with an excess of putrescine in the presenceof pyridine borane. HA or derivatives thereof were purified by repeatedethanol precipitation prior to detection of free amino groups on the HAchain by using the ninhydrin test (Sheng et al., Anal. Biochem. 211,242–249 (1993)).

FIG. 2 shows ¹H NMR of native HA (FIG. 2A) and an HA-derivative withprotected aldehyde functionality (FIG. 2B) in D₂O at 300 Mhz. Peaks areassigned as indicated on the structural formula.

FIG. 3 shows ¹H NMR of HA-derivatives with amine functionality formedfrom putrescine (FIG. 3A), histidine (FIG. 3B), lysine (FIG. 3C), andadipic dihydrazide (FIG. 3D) in D₂O at 300 Mhz. Peaks are assigned asindicated on the structural formula.

FIG. 4 shows digestion of crosslinked HA hydrogels with hyaluronidase.In FIG. 4A, HA-hydrogels were formed by crosslinking 12 mg/ml highlymodified (˜65–70%) HA-amine (adipic dihydrazido-HA) with 15 mg/ml(SPA)₂-PEG. Gels were incubated with different concentrations of bovinetesticular hyaluronidase for the indicated time and the degradation ofthe gels was measured by the release of glucuronic acid into thesupernatant using the carbazole method (Bitter and Muir, Anal Biochem.4, 330–334 (1962)). In FIG. 4B, HA-hydrogels were formed by crosslinking12 mg/ml optimally modified (˜20–25%) HA-amine (adipic dihydrazido-HA)with 15 mg/ml (SPA)₂-PEG (⋄); 12 mg/ml highly modified (˜65–70%) adipicdihydrazido-HA with 15 mg/ml (SPA)₂-PEG (Δ); 12 mg/ml optimally modified(˜20–25%) lysine methylester-HA with either 15 mg/ml (SPA)₂-PEG (Λ) or0.44 mg/ml glutaraldehyde (□), and 12 mg/ml optimally modified (˜10–15%)diaminobutyl-HA with 15 mg/ml (SPA)₂-PEG (o). Gels were incubated withdifferent concentrations of bovine testicular hyaluronidase for theindicated time and the degradation of the gels was measured as in FIG.4A above.

FIG. 5 shows phase contrast images of cells cultured on differentcrosslinked HA hydrogels. FIG. 5A: Dedifferentiated chondrocytescultured on a hydrogel formed from highly modified (˜65–70%) HA-amine(adipic dihydrazido-HA) crosslinked with 5 mg/ml (SPA)₂-PEG aggregate asa consequence of inability to adhere to substratum. FIG. 5B: Cellscultured on a hydrogel made up by the same HA-amine crosslinked with0.25 mg/ml glutaraldehyde show a rounded morphology and no aggregationindicating that they are able to adhere to the substratum. FIG. 5C:Cells cultured on a hydrogel formed from the HA-amine (adipicdihydrazido-HA) modified to a degree of ˜20–25% and crosslinked with 2mg/ml (SPA)₂-PEG adhere to the substratum, spread and subsequentlyinfiltrate the hydrogel. All images show cells 24 h post seeding butmorphology remains the same even after several days in culture.

FIG. 6 shows in vivo evaluation of HA hydrogels formed from different HAderivatives using aldehyde-mediated crosslinking. Subcutaneousimplantation in rats of HA hydrogels consisting of (FIG. 6A) 12 mg/mloptimally modified (˜20–25%) HA-amine (adipic dihydrazido-HA)crosslinked with 0.25 mg/ml glutaraldehyde, (FIG. 6B) 7 mg/ml of thesame HA-amine crosslinked with 7 mg/ml HA-aldehyde (periodate oxidized),(FIG. 6C) 7 mg/ml of the same HA-amine crosslinked with 7 mg/mlHA-aldehyde (deprotected amino-dimethyl acetal-HA, ˜10–15% modified), or(FIG. 6D) 7 mg/ml of the same HA-amine crosslinked with 7 mg/mlHA-aldehyde (deprotected hydrazido-dimethyl acetal-HA, ˜40–45%modified). The hydrogels also contained 1 mg/ml prefibrillized intactcollagen type I, 200 μg/ml BMP-2 and 500 ng/ml IGF-1 to induce boneformation. Tissue specimens were harvested 10 days post implantation,fixed in formalin and processed for histology by paraffin embedding.Sections were stained with Haematoxylin/Eosin. m, matrix material (note:matrix material shrinks during dehydration); s, skin (indicatesorientation of implant).

FIG. 7 shows in vivo evaluation of HA hydrogels crosslinked withdifferent NHS-esters. Subcutaneous implantation in rats of HA hydrogelsconsisting of (FIG. 7A) 12 mg/ml highly modified (˜65–70%) HA-amine(adipic dihydrazido-HA) crosslinked with 15 mg/ml (SPA)₂-PEG, (FIG. 7B)12 mg/ml optimally modified (˜20–25%) HA-amine (adipic dihydrazido-HA)crosslinked with 15 mg/ml SPA₂-PEG, or (FIG. 7C) 12 mg/ml of the sameoptimally modified HA-amine crosslinked with 3 mg/ml DTSSP (crosslinkerconcentrations are equal on a molar basis). The hydrogels also contained1 mg/ml prefibrillized intact collagen type I, 200 μg/ml BMP-2 and 50ng/ml TGF-β2 to induce bone formation. Tissue specimens were harvested10 days post implantation, fixed in formalin and processed for histologyby paraffin embedding. Sections were stained with Haematoxylin/Eosin. m,matrix material (note: matrix material shrinks during dehydration); s,skin (indicates orientation of implant).

FIG. 8 shows differential effect of growth factors incorporated into HAhydrogels on tissue transformation. Subcutaneous implantation in rats ofthe HA hydrogel formed from 12 mg/ml optimally modified (˜20–25%)HA-amine (adipic dihydrazido-HA) crosslinked with 15 mg/ml (SPA)₂-PEG.The hydrogels also contained 1 mg/ml prefibrillized intact collagen typeI, and were supplemented either with 200 μg/ml BMP-2 and 500 ng/ml IGF-1(FIG. 8A), or 200 μg/ml BMP-2 and 50 ng/ml TGF-β2 (FIG. 8B). Tissuespecimens were harvested 10 days post implantation, fixed in formalinand processed for histology by paraffin embedding. Sections were stainedwith Haematoxylin/Eosin.

BRIEF DESCRIPTION OF THE REACTION SCHEMES

Scheme 1 illustrates periodate oxidation of hyaluronic acid.

Scheme 2 illustrates coupling of amines to hyaluronic acid with EDC viaan active triazole ester intermediate.

Scheme 3 illustrates coupling of amines to hyaluronic acid with EDC viaan active N-hydroxysuccinimde ester intermediate.

Scheme 4 illustrates crosslinking of amine functionalized hyaluronicacid with various bifunctional N-hydroxysuccinimde ester crosslinkers toform hydrogels. (1. (SPA)₂-PEG; 2. DTSSP).

Scheme 5 illustrates crosslinking of amine functionalized hyaluronicacid with glutaraldehyde to form hydrogels. In addition to theconventional reaction of aldehydes with amines that results in theformation of a Schiff base, glutaraldehyde is also known to undergopolymerization by aldol condensation yielding polymers withα,β-unsaturated aldehydes at neutral or slightly alkaline pH (Richardsand Knowles, J. Mol. Biol. 37, 231–233 (1968)). Subsequent, nucleophilicaddition of amines at the ethylenyl double bond creates a stablecrosslink.

Scheme 6 illustrates formation of hydrogels with aldehyde functionalizedhyaluronic acid. (1. amine functionalized HA; 2. bifunctional amine)

DETAILED DESCRIPTION OF THE INVENTION

Using the methods of our invention, we generate an activated form of HAthat differs minimally from native HA to conserve its uniquephysico-chemical properties. We also effect a minimal change affectingonly a relatively small number of dissaccharide units of native HA sothat we do not alter its property to serve as a cell substratum.

We initially attempted to generate an aldehyde derivative of HA byreduction of the carboxyl groups of the glucuronic acid moieties intoaldehydes using 9-borabicyclo-3,3-nonane, a method that allows directconversion of the carboxylic acid into the aldehyde (Cha et al., Bull.Korean Chem. Soc. 9, 48–52 (1988), Cha et al., Org. Prep. Proc. Int. 21,451–477 (1989)):HA—COOH(I)------>HA—CHO  (II)

However, even though preliminary testing indicated the conversion of thecarboxyl groups into aldehydes to a degree of approximately 5–10% (FIG.1), mixtures of concentrated, viscous HA-aldehyde solutions (˜10 mg/ml)with ‘small’ polyamines such as putrescine, lysine, polylysine,histidine, or polyhistidine did not generate stable gels in a reasonabletime frame to be suitable for in situ polymerization. It is important tonote that the chemical properties of HA are determined not merely by itsfunctional groups per se but also by the accessibility of thesefunctional groups of HA in an aqueous environment, which is related tothe overall conformational structure and Theological properties of HA.HA behaves like a hydrogel in an aqueous media even in the absence ofcrosslinks because it forms a network stabilized by hydrogen bonds andvan der Waals forces (Laurent and Fraser, supra). To increase theaccessibility of functional groups, we introduced a spacer between thefunctional group and the HA chain.

Introducing a Functionalized Side Chain onto HA

We subsequently developed methodology for introducing side chains intoHA by carbodiimide-mediated coupling of primary or secondary amines tothe carboxyl group of the glucuronic acid moiety using an active esterintermediate. We have used this methodology to generate HA withdifferent terminal functional groups for crosslinking including acetals,aldehydes, amines, and hydrazides. A wide range of functionalized aminesare commercially available which allows us to introduce a wide varietyof different functional groups useful for crosslinking underphysiological conditions using this methodology, including maleimidesthat react specifically with sulfhydryls or arylazides forphotocrosslinking besides the amines and aldehydes described below.

Direct carbodiimide-mediated coupling of amines to the carboxyl group ofHA in an aqueous environment, e.g., with EDC(1-ethyl-3-[3-dimethylaminopropyl] carbodimide), does not yield thepredicted product since the O-acyl isourea that is formed as a reactiveintermediate rearranges rapidly to a stable N-acyl urea (Kuo et al.,supra). We have demonstrated that by “rescuing” the active O-acylisourea by formation of a more hydrolysis resistant and non-rearrangableactive ester intermediate, the coupling of primary amines to HA ispossible. A wide variety of active carboxylic esters exist and could beformed for further reaction including NHS-esters, nitrophenol esters,triazole esters, sulfonic esters, etc., as long as the reagent for theirpreparation is soluble in H₂O or in other polar solvents such asdimethylsulfoxide or dimethylformamide. HA is soluble in H₂O or otheraprotic polar solvents in native form and when prepared as a sodium saltor when prepared as a tetrabutylammonium salt as described in U.S. Pat.No. 4,957,744, respectively. We have formed active esters of HA with1-hydroxybenzotriazole (HOBT) or N-hydroxysulfo-succinimide using theH₂O soluble carbodiimide EDC for coupling. Nucleophilic additon to theester formed from HOBT requires the amine to be presented inunprotonated form at acidic pH (about 5.5 to 7.0). Only a limited numberof amines including hydrazines and activated amines, e.g., ethylenediamine, have pKa values in a suitable range and are consequentlyunprotonated and reactive with the ester-intermediate formed with HOBT(Scheme 2). Simple primary amines, e.g., putrescine, which typicallyhave pKa values >9 do not form significant amounts of adduct underacidic coupling conditions. The N-hydroxysulfosuccinimide-derivedintermediate allows for the coupling reaction to be carried out atneutral pH (about 7.0 to 8.5) and consequently yields products byreaction with simple primary amines (Scheme 3).

Consequently, this methodology allows for the following reactions to becarried out:HA—COOH(I)+H₂N—R(III)------>HA-CO—NH—R  (IV)HA—COOH(I)+R′—NH—R(V)------>HA-CO—NR′—R  (VI)wherein R and R′ are alkyl, aryl, alkylaryl or arylalkyl side chainswhich may contain hetero atoms such as oxygen, nitrogen, and sulfur. Theside chain may be branched or unbranched, and be saturated or maycontain one or more multiple bonds. The carbon atoms of the side chainmay be continuous or may be separated by one or more functional groupssuch as an oxygen atom, a keto group, an amino group, an oxycarbonylgroup, etc. The side chain may be substituted with aryl moieties orhalogen atoms, or may in whole or in part be formed by ring structuressuch as cyclopentyl, cyclohexyl, cycloheptyl, etc. The side chain mayhave a terminal functional group for crosslinking such as aldehyde,amine, arylazide, hydrazide, maleimide, sulfhydryl, etc. The side chainmay be a bioactive peptide, e.g., containing receptor binding sites,crosslinking sites for transglutaminases, or proteolytic cleavage sites.

Terminal functional groups of the side chain useful for crosslinking ofHA under physiological conditions may be selected from the followinglist:

-   1. aldehydes, see Examples    H₂N—R—CHO  (VII)-   2. amines, see Examples    H₂N—R—NH₂  (VIII)-   3. arylazides, e.g., 4-(p-azidosalicylamido)butylamine

-   4. maleimides, e.g.,    4-(N-maleimidomethyl)cyclohexane-1-carboxylhydrazide

-   5. sulfhydryls, e.g., S-methylsulfide cysteine    H₂N—R—SH  (XI)-   6. peptides, e.g., H₂N-APQQEA, comprising substrate sites for    enzymatic crosslinking, e.g., by transglutaminases (Parameswaran et    al., Proc. Natl. Acad. Sci. U.S.A. 87, 8472–8475 (1990); Hohenadl et    al., J. Biol. Chem. 270, 23415–23420 (1995)).

The carbodiimides useful in this reaction may be represented by thefollowing formula:R—N═C═N—R′  (XII)wherein R and R′ comprise side chains of variable structure as describedabove in detail. Carbodiimides which are soluble in an aqueous media arepreferred.

The active ester may be of the following class and be formed bycarbodiimide-mediated coupling of a compound for preparation of theseactive esters known to a person in the art:

-   1. triazole esters, e.g. 1-hydroxybenzotriazole

-   2. NHS-esters, e.g. N-hydroxysulfosuccinimide

-   3. nitrophenol esters, e.g. p-nitrophenol

Preparation of HA-Aldehyde Derivatives

A side chain containing a protected aldehyde in the form of an acetalwas prepared as follows.N-(2,2-dimethoxyethyl)-4-(methoxycarbonyl)butanamide was obtained fromaminoacet-aldehyde dimethyl acetal and mono-methyl succinate using EDCcoupling. An amino group for the coupling to HA was subsequentlyintroduced by reacting the product with hydrazine, yielding the desiredside chain with the protected aldehyde,N-(2,2-dimethoxyethyl)-4-(hydrazido)butanamide. The side chain wascoupled to HA using HOBT and EDC (Scheme 2). An acetal side chain with asimple primary amine, 1-aminoethyl-dimethylacetal, was conjugated to HAusing N-hydroxysuccinimide and EDC (Scheme 3). The HA-derivatives werepurified by ethanol precipitation. The nature of the HA-derivatives wasconfirmed by ¹H NMR (FIG. 2). The HA-acetal derivatives are easilyactivated to the reactive aldehydes by mild acid treatment. OtherHA-aldehyde derivatives with variations in the length of the side chainhave been prepared in a similar manner. See Examples 1–3.

Preparation of HA-amine Derivatives

Diaminoethane, lysine methyl ester, histidine, and adipic, succinic orsuberic dihydrazide was coupled to HA using HOBT and EDC (up to 5-foldexcess depending on the desired degree of modification) and adjustingthe pH to ˜6.5 by repeated addition of 0.1M HCl during the reaction(Scheme 2). HA-derivatives were also prepared in a similar manner usingN-hydroxysulfosuccinimide and primary amines containing unconjugatedamino groups with a higher pKa (>9) such as 1,4-diaminobutane or 1,6diaminohexane (Scheme 3). The HA derivatives were purified by repeatedethanol precipitation and by extensive dialysis, and the nature of theHA derivatives was confirmed by ¹H NMR (FIG. 3). The degree ofmodification was calculated from the NMR data and found to be as high as70%. Reaction conditions were subsequently adjusted such that a degreeof modification of approximately 20% was achieved. Limiting the amountof carbodiimide proved to be most successful in controlling the degreeof modification. A degree of modification of 10–25% yielded efficientcrosslinking but also a molecule that would still be recognized byglycosidases and by HA receptors as HA and thus allow for recognitionand processing of the material by cells (see below). Similar HAderivatives were synthesized using succinic, adipic or subericdihydrazide or diaminoethane, -butane, or -hexane to study the effect ofthe length of the spacer separating the introduced functional group fromthe HA-chain on the subsequent crosslinking. See Examples 4–8.

Crosslinked HA Hydrogels

The functionalized HA molecules can be crosslinked by reacting HAderivatives with different functionalities or using homo- orheterobifunctional crosslinkers which are available in large variety.The following basic reaction schemes are suitable for crosslinking ofthe described forms of modified HA (see Examples 9–12):

-   1: aldehyde-mediated crosslinking

-   2. active ester-mediated crosslinking

-   3. maleimide-mediated cros slinking

-   4. photo-crosslinking

-   5. enzymatic crosslinking (transglutaminase)    R₁—(CH)₂—CONH₂+H₂N—R₂------>R₁—(CH)₂—CO—NH—R₂

Crosslinking of the HA-amine derivatives (Mr˜10⁶) with bifunctionalactive esters, e.g. polyethyleneglycol-bis-succinimidyl-propionate[(SPA)₂-PEG] and reducible 3,3′-dithiobis(sulfo-succinimidyl-propionate)(DTSSP) (Scheme 4), or bifunctional aldehydes, e.g. glutaraldehyde(Scheme 5), generated excellent hydrogels. Stable gels could be formedby crosslinking 5 to 25 mg/ml HA derivative with >0.05 mM aldehydeor >0.2 mM active ester (numbers are reflecting functional groupconcentrations). Optimal gels were generated by crosslinking 10–15 mg/mlHA derivative, modified to a degree of about 10–25%, with about 0.2 mMaldehyde or 0.6 mM active ester. Similarly, crosslinking of theHA-aldehyde derivatives (Mr˜10⁶) (optimally about 10–15 mg/ml) withbifunctional amines (optimally about 0.2 mM) yielded excellent gels(Scheme 6). Conjugated amines such as dihydrazines or benzylamines arerequired for in situ polymerization of HA in this case to resonancestabilize the instable Schiff base product formed by reaction of analdehyde with a primary amine (i.e. hydrazines yield a more stablehydrazone linkage). Hydrogels were also formed from an equimolar mixtureof HA-aldehyde derivatives and the different HA-amine derivatives(Scheme 6). Optimal gels were formed with ˜15 mg/ml of the HAderivatives. At the optimal concentrations of HA and crosslinker,gelation occurred typically in about 30 sec. to 5 min. which is suitablefor in situ polymerization. The crosslinked HA hydrogels were sensitiveto glycosidases, i.e. testicular hyaluronidase, indicating that they arebiodegradable (FIG. 4).

A number of different tests including cell culture assays and animalexperiments served to assess biocompatibilty of the formulatedbiomaterials. Embedding of chondrocytes into the polymerizing HAhydrogels showed that neither aldehyde nor NHS-ester-mediatedcrosslinking was toxic to cells at the concentrations employed. Seedingof cells on top of prepolymerized HA hydrogels showed a wide variety ofcellular behaviours depending on the nature of the crosslinker andcrosslinking density (FIG. 5). Highly crosslinked HA hydrogels wereimpenetrable to cells (FIGS. 5, A and B), while optimally crosslinkedgels were infiltrated (FIG. 5C). Supplementation of the HA hydrogelswith cell adhesion molecules such as fibronectin (in the range of 0.1 to1 mg/ml) did induce adhesion and spreading of cells on the materialsindependent of the nature of the crosslinker and the crosslinkingdensity, but did not change the results with regard to cellinfiltration, demonstrating that the lack of infiltration is due to thehigh crosslinking density and not the absence of cell-matrixinteractions. See below and FIG. 7.

Subcutaneous implantation of biomaterials in rats is the establishedmodel for evaluation of biocompatibility of biomaterials (Laurencin etal., J. Biomed. Mat. Res. 24, 1463–1481 (1990)) and for induction ofectopic bone formation by members of the TGF-β gene family, and bonemorphogenetic proteins (BMP) in particular (Wang et al., Proc. Natl.Acad. Sci. U.S.A. 87, 2220–2224 (1990); Sampath et al., J. Biol. Chem.267, 20352–20362 (1992)). Taking into consideration the cell cultureresults, we have formulated a number of HA hydrogels for in vivobiocompatibility testing in this model. Implantation of prepolymerizedHA hydrogel discs loaded with recombinant BMP-2 and IGF-1 or TGF-β2subcutaneously in rats showed a mild fibrosis with a varying degree ofcartilage and bone formation depending on the nature of the HAbiomaterial (FIGS. 6 and 7). The growth factors were mixed with the HAderivatives prior to gelling and the induction of bone formationsuggests that neither reaction mechanism used for HA crosslinking(aldehyde or active ester-mediated reactions) significantly affected thebiological activity of the growth factors. Little inflammation wasobserved with active ester crosslinked HA-amine derivatives (FIG. 7) orwith HA-amine derivatives crosslinked with various HA-aldehydederivatives (FIGS. 6B–6D) while a stimulation of foreign body giantcells was seen when the same HA-amine derivatives were crosslinked withglutaraldehyde (FIG. 6A). The degree of modification of HA stronglyaffected the resorption and transformation rate of the biomaterials(FIGS. 7A, 7B). Nevertheless, limited bone formation was seen even witha biomaterial formed from a highly modified (65–70%) HA-amine derivative(FIG. 7A). The absence of bone formation with a smaller bifunctionalNHS-ester crosslinker indicates that the size of the generatedcrossbridge is crucial for resorption and cellular infiltration (FIG.7C). This is probably due to the difference in pore size of the materialformed with crosslinkers of different sizes. The infiltration andtransformation rate was similar with BMP-2/IGF-1 and BMP-2/TGF-β2 loadedbiomaterials, indicating that the resorption rate is a materialproperty. However, at ten days post-implantation, the newly formedtissue was largely cartilage in the first group and largely bone in thesecond group (FIG. 8), exemplifying the angiogenic effect of TGF-β2(Yang and Moses, J. Cell. Biol., 111, 731–741 (1990)). This demonstratesthat the biological activity of the HA material can be modulated byinclusion of different bioactive factors. The lack of significantadverse effects and the demonstration of the desired biological activityof these novel HA biomaterials in vivo demonstrates their usefulness asa delivery vehicle for cells and growth factors in the field of tissueregeneration.

There are several approaches to the production of HA, includingextraction from tissue and biosynthesis. Extraction from tissuetypically uses fresh or frozen cocks' combs (U.S. Pat. No. 5,336,767),although other tissues including the synovial fluid ofjoints (Kvam etal., Anal. Biochem. 211, 44–49 (1993)), human umbilical cord tissue,bovine vitreous humor, and bovine tracheae, have been used. It is alsopossible to prepare HA by microbiological methods, such as bycultivating a microorganism belonging to the genus Streptococcus whichis anhemolytic and capable of producing HA in a culture medium (U.S.Pat. Nos. 4,897,349; 4,801,539; 4,780,414; 4,517,295; 5,316,926). The HAraw material for preparing the compositions of the invention preferablyconsists of high molecular weight HA, more preferably of molecularweight greater than 0.5 million daltons, and more preferably ofmolecular weight greater than one million daltons. The HA raw materialfor the compositions of examples of this invention described herein wasobtained from Genzyme Corp. (Cambridge, Mass.), and had a molecularweight greater than one million daltons. The size of the HA wasunchanged after derivatization.

The compositions of the invention have many therapeutic uses. The factthat the compositions may be cured in a surgically practical time frameof one to five minutes in situ with concurrent crosslinking to thetissue surfaces allows for employment as a tissue glue. Many situationsin various surgical applications require such adhesives. For example,the compositions of the invention may be used to stem hemorrhage ingeneral surgery, reconstruct nerves and vessels in reconstructive,neuro- and plastic surgery, and to anchor skin, vascular, or cartilagetransplants or grafts in orthopedic, vascular, and plastic surgery.Those of skill in the art may choose and design particular embodimentsof the invention which are particularly suitable for a desiredapplication, by adjusting several factors, including: (1) the degree offunctionalization of HA, which affects the crosslinking density of thematerial and interaction with cellular proteins, including receptors andglycosidases; (2) the concentration of the crosslinker, which affectsthe crosslinking density of the material; (3) the size of the generatedcross-bridge, which affects the pore size of the material; (4) thenature of the crosslinking mechanism, which determines polymerizationtime and the specificity of the reaction; and (5) the nature of thecross-bridge, which provides biological cues. See FIGS. 4, 5, and 7 fordata concerning HA hydrogels with different crosslinking densities andpore sizes. Generally, active ester- or photo-crosslinking are preferredto form materials for applications requiring fast gelation and strongbonding with tissue surfaces, such as tissue glues. Materials withanti-adhesive properties, which are useful to form tissue separations orfor tissue augmentation, are formed from highly modified HA derivativeswith low molecular weight crossilinkers, which generates a densematerial with very small pores, thereby minimizing cell adhesion andinfiltration. Conversely, biodegradable scaffolds for tissue repair areformed from HA with a limited degree of derivativization and highmolecular weight crosslinkers, which generate a porous, biodegradablematerial. The crossbridge may even contain biological cues, such aspeptide sequences, which facilitate material degradation by, forexample, proteolysis or cellular infiltration (e.g., the RGD sequence).

Compositions of this invention were designed to serve as a vehicle forthe delivery of cells or bioactive molecules such as growth factors tostimulate focal repair. The crosslinked HA derivatives are poroushydrogels in which biologically or therapeutically active compounds(e.g., growth factors, cytokines, drugs, and the like) can be physicallyor chemically incorporated. These compounds will then be subject tosustained release by chemical, enzymatic, and physical erosion of thehydrogel and/or the covalent linkage between the HA chain andbiologically active compound over a period of time. Local delivery ofgrowth factors with such a scaffold facilitates wound healing and tissueregeneration in many situations. For example, the compositions of theinvention may be used not only to promote bone formation and stimulatecartilage repair in orthopedic procedures, as described more fullybelow, but also to treat pathological wound conditions such as chroniculcers. They may also serve as a scaffold to generate artificialtissues, e.g., cartilage (Hauselmann et al., Am. J. Physiol. 271,C742–752 (1996)), through proliferation of autologous cells in culture.Similar procedures for generation of equivalents of other tissues ororgans, including skin, liver, and others, in culture may be developedin the future and may be used in combination with the compositions ofthe invention.

Highly crosslinked materials have an anti-adhesive property with respectto cells, and such compositions may be used to generate tissueseparations and to prevent adhesions following surgery. See FIGS. 5A and7C, showing highly modified HA-amine, i.e., adipic dihyrazido HA,preferably crosslinked with low molecular weight bifunctional NHS-ester.The viscoelastic properties of HA make it particularly well suited forthis purpose, and it is used clinically to achieve temporal pain reliefby repeated intraarticular injections in arthropathies as a “jointlubricant”, and as a protective agent for eye irritations and inophthalmic surgery. The technique of tissue separation is used in facialreconstruction in plastic surgery and dentistry. Prevention of theformation of adhesions is particularly relevant in reconstructivesurgery of tendons, in surgical procedures in the urogenital system, andin thoracic surgery. Many different HA-based materials are already inclinical use in these areas. (See products manufactured by AnikaTherapeutics, Inc. (Woburn, Mass.), Biomatrix, Inc. (Ridgefield, N.J.),Genzyme Corp. (Cambridge, Mass.), and Fidia, S.p.A. (Abano Terme,Italy)). Those of skill in the art may choose and design particularembodiments of the invention which are particularly suitable for adesired application by selecting distinct features as outlined above.

The injectable nature of the compositions of the invention also rendersthem suitable for tissue augmentation in plastic surgery, where the HAmatrix serves primarily as a biocompatible filler material, e.g., forfilling dermal creases or lip reconstruction. Again, those of skill inthe art may choose and design particular embodiments of the inventionwhich are particularly suitable for a desired application, as outlinedabove.

The half-life of pharmacological compounds, both synthetic andbiological, has been shown to be drastically increased when delivered ina form conjugated to HA (Larsen and Balazs, Adv. Drug Delivery Rev. 7,279–293 (1991); Drobnik, J., Drug Delivery Rev. 7, 295–308 (1991)). Thefunctionalized forms of HA provided by this invention allow for easysubstitution with pharmacologically active agents, such asanti-inflammatories, analgesics, steroids, cardiovascular agents,anti-tumor agents, immunosuppressants, sedatives, anti-bacterial,anti-fungal, and anti-viral agents, etc., and may be used for sustaineddrug release over time, either locally in hydrogel form or systemicallyin free form.

In orthopedic surgery, the functionalized forms of HA of this inventionhave applications as a tissue glue or bioactive matrix material in thetreatment of chondral and osteochondral fractures, osteochondritisdissecans, meniscal tears, as well as ruptured ligaments, tendons, ormyotendineous junctions. The HA materials of this invention may serve tofacilitate anchorage of chondral or osteochondral transplants or grafts,or other biological or artificial implant materials, or to stimulate newbone or cartilage formation by serving as a scaffold for cells or as adelivery vehicle for growth factors. One general approach to promotearticular cartilage repair based on the compositions of the inventioncomprises using: (1) in situ polymerized HA hydrogel as a matrix to fillthe defect which is to be repaired and to provide a scaffold for repaircells, (2) an optional chemotactic agent to attract repair cells to thematrix and defect site, or alternatively, autologous chondrocytes ormesenchymal stem cells, (3) an optional factor to promote cellularproliferation of repair cells in the matrix and defect site; (4)sustained release of a transforming factor by the HA hydrogel over timeto promote differentiation of the repair cells into chondrocytes whichproduce new cartilage; and (5) an optional anti-angiogenic factor toprevent vascularization and endochondral ossification of the newlyformred cartilage. Examples of suitable factors are known to thoseskilled in the art, and may be found in, e.g., U.S. Pat. No. 5,368,858.

Delivery of growth factors in active form may require supplementation ofthe HA hydrogels with additional ingredients, such as growth factorbinding molecules like heparin and collagen. For example, for cartilagerepair, crosslinked hyaluronic acid hydrogels that are rapidlyinfiltrated by cells such as those formed from an HA-amine derivativecrosslinked with a polyvalent high molecular weight NHS-estercrosslinker, e.g., (SPA)₂-PEG, are selected which are resorbed andreplaced by repair tissue within about 2 to 3 weeks. In some cases,cells and/or growth factors may be mixed in prior to gelling.

The following are illustrative examples, which are not intended to limitthe scope of the present invention.

EXAMPLES Example 1

Preparation of N-(2,2-dimethoxyethyl)-4-(methoxycarbonyl)butanamide(1)—EDC (4.98 g, 0.026 mol) was added to a mixture of aminoacetaldehydedimethyl acetal (2.18 ml, 20 mmol) and methyl monoester of succinic acid(2.64 g, 20 mmol) in 75 ml of dichloromethane, and the reaction mixturestirred for 24 h at room temperature. The solution was extractedsuccessively with 50 ml each of ice cold solutions of 0.75M sulfuricacid, 1M NaCl, saturated sodium bicarbonate, and 1M NaCl. The organicphase was collected and dried with sodium sulfate. The solvent wasevaporated under reduced pressure yielding a syrup, which showed asingle spot on charring upon TLC in solvent A (R_(f)0.75) and solvent B(R_(f)0.24). The apparent yield of 1 was 65%. ¹H NMR in CDCl₃ δ 5.70(bs, 1H, NH), 4.34 [t, 1H, CH—(OCH₃), 3.67 (s, 3H, COOCH₃), 3.43–3.35 (sand t, 8H, CH₃OC and CHCH₂NH), 2.38–2.26 (m, 4H, CH₂CO).

Formation of Acyl-hydrazide (2) from 1—Anhydrous hydrazine (248 μl, 7.9mmol) was added to a solution of 1 (1.73 g, 7.9 mmol) in 5 ml ofanhydrous methanol. The mixture was stirred at room temperatureovernight and the solvent subsequently evaporated under reduced pressureyielding a solid residue. The residue was dissolved in H₂O (6 ml) andextracted three times with an equal volume of dichloromethane. Theaqueous solution was evaporated to dryness under reduced pressure andthen further dried overnight in vacuo. The crystaline solid (1.04 g, 82%yield) was homogeneous on TLC in solvent A (R_(f)0.10) when visualizedby charring. The ¹H NMR spectrum indicated the loss of the ester methoxygroup when compared to 1.

Preparation of Hydrazido-dimethyl acetal-HA (formula XIX)—Sodiumhyaluronate (100 mg, 0.25 mmol) andN-(2,2-dimethoxyethyl)-4-(hydrazido)butanamide (2) (1.646 g, 7.5 mmol)was dissolved in H₂O (40 ml, 2.5 mg/ml HA). The pH was adjusted to 6.5and HOBT (169 mg, 1.25 mmol) predissolved in a 1:1 mixture of water andDMSO (1 ml) and EDC (240 mg, 1.25 mmol) was added and the reactionmixture was stirred overnight. The pH was subsequently adjusted to 7.0with 1M NaOH and NaCl added to produce a 5% w/v solution. HA wasprecipitated by addition of three volume equivalents of ethanol. Theprecipitate was redissolved in H₂O at a concentration of approximately 5mg/ml and the precipitation repeated twice. The purified product wasfreeze dried and kept at 4° C. under N₂. See FIG. 2B for NMR data of theproduct.

Example 2

Preparation of Aminoacetaldehyde-dimethyl acetal-HA (formula XX)—Sodiumhyaluronate (100 mg, 0.25 mmol) and 2,2-dimethoxyethylamine (0.788 g,7.5 mmol) was dissolved in H₂O (40 ml, 2.5 mg/ml HA). The pH wasadjusted to 7.5 and NHS.SO₃Na (268 mg, 1.25 mmol) and EDC (240 mg, 1.25mmol) was added and the reaction mixture was stirred overnight. The pHwas subsequently adjusted to 7.0 with 1M NaOH and NaCl added to producea 5% w/v solution. HA was precipitated by addition of three volumeequivalents of ethanol. The precipitate was redissolved in H₂O at aconcentration of approximately 5 mg/ml and the precipitation repeatedtwice. The purified product was freeze dried and kept at 4° C. under N₂.

Example 3

Deprotection of HA-acetals to form HA-aldehydes—The acetal modifiedHA(formula XXI) was dissolved in H₂O to a concentration of 5–10 mg/mland 1M HCl was added to give a final concentration of 0.025M. Thesolution was then allowed to stand at room temperature for 0.5 to 1.0 h.The solution was neutralized by the addition of 1M NaOH, yielding thedeprotected HA-aldehyde (formula XXII).HA—CO—R—CH(OCH₃)₂(XXI)------>HA-CO—R—CHO  (XXII)

Example 4

Preparation of Diaminoethane-HA (formula XXIII)—Sodium hyaluronate (100mg, 0.25 mmol) and 1,2-diaminoethane HCl (0.998 g, 7.5 mmol) wasdissolved in H₂O (40 ml, 2.5 mg/ml HA). The pH was adjusted to 6.5 andHOBT (169 mg, 1.25 mmol) predissolved in a 1:1 mixture of water and DMSO(1 ml) and EDC (240 mg, 1.25 mmol) was added and the reaction mixturewas stirred overnight. The pH was subsequently adjusted to 7.0 with 1MNaOH and NaCl added to produce a 5% w/v solution. HA was precipitated byaddition of three volume equivalents of ethanol. The precipitate wasredissolved in H₂O at a concentration of approximately 5 mg/ml and theprecipitation repeated twice. The purified product was freeze dried andkept at 4° C. under N₂.

Example 5

Preparation of L-Lysine methyl ester-HA (formula XXIV)—Sodiumhyaluronate (100 mg, 0.25 mmol) and L-lysine methyl esterdihydrochloride (1.748 g, 7.5 mmol) was dissolved in H₂O (40 ml, 2.5mg/ml HA). The pH was adjusted to 6.5 and HOBT (169 mg, 1.25 mmol)predissolved in a 1:1 mixture of water and DMSO (1 ml) and EDC (240 mg,1.25 mmol) was added and the reaction mixture was stirred overnight. ThepH was subsequently adjusted to 7.0 with 1M NaOH and NaCl added toproduce a 5% w/v solution. HA was precipitated by addition of threevolume equivalents of ethanol. The precipitate was redissolved in H₂O ata concentration of approximately 5 mg/ml and the precipitation repeatedtwice. The purified product was freeze dried and kept at 4° C. under N₂.See FIG. 3C for NMR data of the product.

Example 6

Preparation of L-Histidine methyl ester HA (formula XXV)—Sodiumhyaluronate (100 mg, 0.25 mmol) and L-histidine methyl esterdihydrochloride (1.815 g, 7.5 mmol) was dissolved in H₂O (40 ml, 2.5mg/ml HA). The pH was adjusted to 6.5 and HOBT (169 mg, 1.25 mmol)predissolved in a 1:1 mixture of H₂O and DMSO (1 ml) and EDC (240 mg,1.25 mmol) was added and the reaction mixture was stirred overnight. ThepH was subsequently adjusted to 7.0 with 1M NaOH and NaCl added toproduce a 5% w/v solution. HA was precipitated by addition of threevolume equivalents of ethanol. The precipitate was redissolved in H₂O ata concentration of approximately 5 mg/ml and the precipitation repeatedtwice. The purified product was freeze dried and kept at 4° C. under N₂.See FIG. 3B for NMR data of the product.

Example 7

Preparation of Hydrazido-HA (formula XXVI)—Sodium hyaluronate (100 mg,0.25 mmol) and dihydrazide i.e. adipic dihydrazide (1.31 g, 7.5 mmol)was dissolved in H₂O (40 ml, 2.5 mg/ml HA). The pH was adjusted to 6.5and HOBT (169 mg, 1.25 mmol) predissolved in a 1:1 mixture of water andDMSO (1 ml) and EDC (240 mg, 1.25 mmol) was added and the reactionmixture was stirred overnight. The pH was subsequently adjusted to 7.0with 1M NaOH and NaCl added to produce a 5% w/v solution. HA wasprecipitated by addition of three volume equivalents of ethanol. Theprecipitate was redissolved in H₂O at a concentration of approximately 5mg/ml and the precipitation repeated twice. The purified product wasfreeze dried and kept at 4° C. under N₂. See FIG. 3D for NMR data of theproduct.

Example 8

Preparation of Diaminoalkyl-HA (formula XXVII)—Sodium hyaluronate (100mg, 0.25 mmol) and a diaminoalkane, i.e. 1,2-diaminobutane HCl (1.208 g,7.5 mmol) was dissolved in H₂O (40 ml, 2.5 mg/ml HA). The pH wasadjusted to 7.5 and NHS.SO₃Na (268 mg, 1.25 mmol) and EDC (240 mg, 1.25mmol) was added and the reaction mixture was stirred overnight. The pHwas subsequently adjusted to 7.0 with 1M NaOH and NaCl added to producea 5% w/v solution. HA was precipitated by addition of three volumeequivalents of ethanol. The precipitate was redissolved in H₂O at aconcentration of approximately 5 mg/ml and the precipitation repeatedtwice. The purified product was freeze dried and kept at 4° C. under N₂.See FIG. 3A for NMR data of the product.

Example 9

Formation of crosslinked HA hydrogels—The general procedure for formingcrosslinked HA hydrogels is as follows: Modified HA is dissolved byagitation in H₂O or phosphate buffered saline (pH 7.4–8.5) at aconcentration of 5–25 mg/ml. The degree of modification of the HAderivative is derived from the integration of the ¹H NMR peaks. Aftercomplete dissolution, the HA derivative solution is transferred to a 1ml syringe. When reacting the HA derivatives with low molecular weightcrosslinkers, a slight excess of the compound (about 1.1 molarequivalent of functional groups) is dissolved in a second 1 ml syringein 1/10 of the HA derivative volume immediately prior to use. Thesyringes are connected while paying special attention to excluded air,the contents are rapidly mixed, typically with 20 passages, and thenextruded. When reacting HA derivative molecules with differentfunctionalities, 0.5–1.0 equivalent of HA-aldehyde is mixed with 1equivalent of HA-hydrazine, depending on the degree of modification ofthe HA derivatives. At room temperature, gelation occurs within about 30seconds to several minutes, depending on the formulation, and the gelproperties do not significantly change after approximately 5 minutes.

Example 10

Digestion of crosslinked HA hydrogels with hyaluronidase—The generalprocedure for digestion of crosslinked HA hydrogels is as follows: HAhydrogels are formed in 1 mL syringes by crosslinking 12 mg/ml HA-aminein phosphate buffered saline with various crosslinkers as indicated inFIG. 4. Gelling is allowed to occur for 1 hour at 37° C. for thereaction to be complete, after which identical ˜100 μl cylindrical gelsare formed by cutting the syringes with a razor blade. The gels areincubated with different concentrations of bovine testicularhyaluronidase (Sigma) 50–5000 U/mL in 400 μl of 30 mM citric acid, 150mM Na₂HPO₄, pH 6.3, 150 mM NaCl for the indicated time 0–48 hours.Degradation of the gels is determined from the release of glucuronicacid into the supernatant as measured using the carbazole method (Bitterand Muir, supra). See FIG. 4.

Example 11

Crosslinked HA hydrogels as a matrix for cell culture—Chondrocytes wereisolated from bovine nose cartilage according to established procedures(Häuselmann et al., Matrix 12, 116–129 (1992; Küttner et al., J. CellBiol. 93, 743–750 (1982)), cultured Ham's F12 medium containing 5% fetalbovine serum and antibiotics, and dedifferentiated by monolayer cultureon plastic. For cytotoxicity studies, cells (2.5×10⁵) were embedded intothe HA hydrogels by gently mixing the trypsinized cells (about 50 to 100μl) with the polymerizing HA and crosslinker mixture (approximately 400μl gel volume) prior to complete setting. Agarose embedded cells servedas a control. After adaptation to the culture conditions (24 h), cellproliferation and metabolic activity was assessed by pulse labeling with[³H]thymidine and [³⁵S]methionine. For cell infiltration studies, HAhydrogels were polymerized in 24-well plates (˜15 mm diameter and 3 mmheight) for 1 h at room temperature, and extensively rinsed withphosphate buffered saline. Cell adhesion molecules or chemotacticfactors, e.g. IGF-1, were added to the HA solution prior to crosslinkingwhen desired. After 24 h, cells (2.5×10⁵) were seeded on top of theHA-hydrogels and cultured as above. At different time points postseeding, gels were fixed in phosphate buffered 4% paraformaldehyde andprocessed for paraffin embedding. Cell infiltration was assessed bystaining sections with Haematoxylin/Eosin. See FIG. 5.

Example 12

Subcutaneous implantation of HA hydrogels in rats—Rats (2–3 per testmaterial) were anaesthetized with ketamine/xylazine, the ventral thoraxand abdomen shaved, and prepared aseptically. A small vertical incisionwas made on either side of the xiphoid cartilage of the sternum and theskin undermined with a blunt instrument to separate the skin from theunderlying tissue. HA hydrogels were polymerized in 3 ml syringes asdescribed. For induction of chondro-osseous differentiation, 1 mg/mlprefibrillized intact collagen type I (Organogenesis, Canton, Mass.),200 μg/ml recombinant BMP-2 (Genetics Institute, Cambridge, Mass.), and500 ng/ml IGF-1 (Celtrix Pharmaceuticals, Santa Clara, Calif.) or 50ng/ml TGF-β2 (Celtrix Pharmaceuticals, Santa Clara, Calif.) were mixedwith the HA solution prior to crosslinking. Collagen fibrils wereprepared by slow polymerization (from dilute solutions of 2–3 mg/ml) ofacid-solubilized collagen in phosphate buffered saline and harvested bycentrifugation following standard protocols (McPherson et al., CollagenRel. Res. 5, 119–135 (1985)). Gelling of the HA hydrogels was allowed tooccur for 24 h at room temperature for the reaction to be complete,after which identical ˜3 mm thick cylindrical gels were prepared bycutting the syringes with a razor blade. HA hydrogel discs were thenplaced in each pocket and the skin incisions closed with sutures. Tendays post operatively, the rats were euthanized and the appearance ofthe implant sites, i.e. degree of inflammation, grossly examined andtissue specimens harvested and processed for histology by fixation inphosphate buffered formalin and paraffin embedding. Sections werestained with Haematoxylin/Eosin and with Safranin-O/fast green, and cellinfiltration and transformation (cartilage and bone formation) inducedby the biomaterial as well as signs of fibrosis and inflammation in thesurrounding tissue evaluated. See FIGS. 6–8.

1. A method for making a derivative of hyaluronic acid (HA), comprisingthe steps of: a) forming an activated ester at a carboxylate of aglucuronic acid moiety of hyaluronic acid; b) substituting at thecarbonyl carbon of the activated ester formed in step (a), a side chaincomprising a nucleophilic portion and a functional group portion; and c)forming a cross-linked hydrogel from the functional group portion of thehyaluronic acid derivative in solution under physiological conditionswherein the forming of a cross-linked hydrogel is not byphoto-cross-linking.
 2. The method of claim 1, wherein the nucleophilicportion is selected from the group consisting of primary amine, andsecondary amine.
 3. The method of claim 1, wherein the functional groupportion is selected from the group consisting of active ester, aldehyde,amine, hydrazide, maleimide, sulfhydryl, and peptide.
 4. The method ofclaim 1, wherein step (a) is performed with an active ester selectedfrom the group consisting of a substituted triazole, N-sulfosuccinimide,nitrophenol, partially halogenated phenol, perhalophenol,pentafluorophenol, HOBT, and NHS, by carbodiimide-mediated coupling. 5.The method of claim 1, wherein step (c) is performed in situ in apatient in need of tissue repair.
 6. A method for forming a matrix for atemporary scaffold for tissue repair according to the method of claim 1,wherein a crosslinker is used in step c), and wherein the crosslinker isselected from the group consisting of polyvalent active ester, aldehyde,amine, maleimide, and sulihydryl.
 7. A method for forming a matrix for atemporary scaffold for tissue repair according to the method of claim 1,wherein the HA derivative comprises a peptide substrate fortransglutaminase, and wherein the HA derivative is crosslinked usingtransglutaminase.
 8. The method of claim 1, wherein step (c) isperformed in the presence of cells.
 9. The method of claim 1, whereinstep (c) is performed in the presence of at least one member selectedfrom the group consisting of growth factors, cytokines, drugs, andbioactive peptides.
 10. The method of claim 9, wherein the bioactivepeptide RGD is present.
 11. The method of claim 9, wherein a bioactivepeptide is present and is a substrate for transglutaminase.
 12. Themethod of claim 11, wherein the bioactive peptide APQQEA is present. 13.The method of claim 11, wherein the growth factor TGF-β or BMP ispresent.